1. Field of the Invention
The present invention relates to a novel hybridoma, a method for generating the same and monoclonal antibodies specific to N-acetylneuraminic acid in the beta-arrangement, which are produced by the hybridoma.
2. Description of the Prior Art
In 1975, Kohler and Millstein developed a method for producing monoclonal antibodies utilizing a hybridoma which makes it possible to almost perpetually produce such an antibody having uniform specificity and other characteristics. Earlier than this, it has been recognized that ordinary anti-sera produced by immunizing an animal with an antigen inclusive of tumour cells contain a variety of antibodies differing in their specificity and characteristic properties. The method by Kohler et al. requires the fusion of spleen cells of an immunized animal with an immortal myeloma cell line and the screening of hybridomas generated by the cell fusion for desired antibody production, i.e., the selection of a clone which can produce or secrete an antibody having a desired specificity from fused cells referred to as "hybridoma" thus generated. Such a clone can continuously produce an antibody inherent thereto. The cloned hybridoma can perpetually be cultured and on the contrary, it can be stored in the frozen state in liquid nitrogen. Thus, the stable supply of individual antibodies can be ensured.
Antibodies are proteins capable of specifically recognizing other molecules or substances known as antigens and also capable of binding to the same. A monoclonal antibody is of course an antibody, but the characteristic properties thereof are quite uniform and it recognizes only one antigen or antigenic determinant. A variety of hybridomas are generated by cell fusion technique, but all of the cloned hybridomas formed by fusing, for instance, myeloma cells with antibody-producing cells derived from an immunized animal are not specific to a desired antigen. Moreover, since antibodies produced by different cloned hybridomas can react with different antigenic determinants on the same molecule, an antibody produced by a specific cloned hybridoma is different from those produced by other clones even if they are specific to a desired antigen. Therefore, it is impossible to correctly forecast the specific site of an antigen molecule which can be recognized by each individual monoclonal antibody. Nowadays, various techniques for producing monoclonal antibodies and those for generating hybridomas capable of producing the same are well known in the art. In this respect, reference can be made to a recent publication "Monoclonal Hybridoma Antibodies: Techniques and Applications", edited by John G. Hurrell, 1983.
It is known that N-acetylneuraminic acid is present at the terminus of a sugar chain of a glycoconjugate (complex carbohydrate) and plays various biologically important roles. It has recently been reported that the substantial increase in the content of free N-acetylneuraminic acid in the blood or urine is observed in patients suffering from malignant tumour or acute inflammatory diseases and thus the quantitative and/or qualitative measurement thereof has attracted special interest recently.
In other words, since the monoclonal antibody has high specificity to a specific antigen and thus detects the same in a high sensitivity, to apply such a monoclonal antibody to the diagnosis of cancer for the purposes of early diagnosis or early treatment thereof would be expected. The diagnosis would be realized by detecting N-acetylneuraminic acid present in, for instance, the serum of individual using a monoclonal antibody as a cancer marker, if such a monoclonal antibody specific to aforesaid free N-acetylneuraminic acid can be obtained.
Under such circumstances, there has been proposed a variety of methods for detecting or quantitatively determining the free N-acetylneuraminic acid in a high sensitivity. However, these methods conventionally established suffer from problems concerning simplicity and specificity to a desired antigen or antigenic determinant.
All the N-Acetylneuraminic acid residues present at the termini of sugar chains of naturally occurring complex carbohydrate are in the alpha-arrangement and on the contrary, the free N-acetylneuraminic acid is in the beta-arrangement. Among the monoclonal antibodies specific to these complex carbohydrates, there are obtained many monoclonal antibodies specifically recognizing sites which contain N-acetylneuraminic acid residues as the epitope, but for the above reasons every antibody does not have an ability of recognizing such free N-acetylneuraminic acid and ability of binding thereto. Moreover, N-acetylneuraminic acid is a hapten with a low molecular weight and thus it has only a low antigenicity. Therefore, it seems very difficult to obtain monoclonal antibodies even if an animal is immunized with such a low molecular weight hapten.